2 results
Search Results
Now showing 1 - 2 of 2
- ItemDirected Evolution of P450 BM3 towards Functionalization of Aromatic O-Heterocycles(Basel : Molecular Diversity Preservation International (MDPI), 2019) Santos, Gustavo de Almeida; Dhoke, Gaurao V.; Davari, Mehdi D.; Ruff, Anna JoĆ«lle; Schwaneberg, UlrichThe O-heterocycles, benzo-1,4-dioxane, phthalan, isochroman, 2,3-dihydrobenzofuran, benzofuran, and dibenzofuran are important building blocks with considerable medical application for the production of pharmaceuticals. Cytochrome P450 monooxygenase (P450) Bacillus megaterium 3 (BM3) wild type (WT) from Bacillus megaterium has low to no conversion of the six O-heterocycles. Screening of in-house libraries for active variants yielded P450 BM3 CM1 (R255P/P329H), which was subjected to directed evolution and site saturation mutagenesis of four positions. The latter led to the identification of position R255, which when introduced in the P450 BM3 WT, outperformed all other variants. The initial oxidation rate of nicotinamide adenine dinucleotide phosphate (NADPH) consumption increased ā140-fold (WT: 8.3 Ā± 1.3 minā1; R255L: 1168 Ā± 163 minā1), total turnover number (TTN) increased ā21-fold (WT: 40 Ā± 3; R255L: 860 Ā± 15), and coupling efficiency, ā2.9-fold (WT: 8.8 Ā± 0.1%; R255L: 25.7 Ā± 1.0%). Computational analysis showed that substitution R255L (distant from the heme-cofactor) does not have the salt bridge formed with D217 in WT, which introduces flexibility into the I-helix and leads to a heme rearrangement allowing for efficient hydroxylation.
- ItemDisulfide Bond Engineering of an Endoglucanase from Penicillium verruculosum to Improve Its Thermostability(Basel : Molecular Diversity Preservation International (MDPI), 2019) Bashirova, Anna; Pramanik, Subrata; Volkov, Pavel; Rozhkova, Aleksandra; Nemashkalov, Vitaly; Zorov, Ivan; Gusakov, Alexander; Sinitsyn, Arkady; Schwaneberg, Ulrich; Davari, Mehdi D.Endoglucanases (EGLs) are important components of multienzyme cocktails used in the production of a wide variety of fine and bulk chemicals from lignocellulosic feedstocks. However, a low thermostability and the loss of catalytic performance of EGLs at industrially required temperatures limit their commercial applications. A structure-based disulfide bond (DSB) engineering was carried out in order to improve the thermostability of EGLII from Penicillium verruculosum. Based on in silico prediction, two improved enzyme variants, S127C-A165C (DSB2) and Y171C-L201C (DSB3), were obtained. Both engineered enzymes displayed a 15ā21% increase in specific activity against carboxymethylcellulose and Ī²-glucan compared to the wild-type EGLII (EGLII-wt). After incubation at 70 Ā°C for 2 h, they retained 52ā58% of their activity, while EGLII-wt retained only 38% of its activity. At 80 Ā°C, the enzyme-engineered forms retained 15ā22% of their activity after 2 h, whereas EGLII-wt was completely inactivated after the same incubation time. Molecular dynamics simulations revealed that the introduced DSB rigidified a global structure of DSB2 and DSB3 variants, thus enhancing their thermostability. In conclusion, this work provides an insight into DSB protein engineering as a potential rational design strategy that might be applicable for improving the stability of other enzymes for industrial applications.